Figure 3.

Differentiation of pluripotent spheres into midbrain progenitor organoids

(A) Differentiation schematic from pluripotent spheres into mNPCs and midbrain organoids containing mature DNs.

(B) Time course bright-field images of WA01 TH midbrain organoids on D0, D5, D10, D15, D20, and D30. Scale bars: 500 μm.

(C) Time course quantification of the images in B showing increase in WA01 TH midbrain organoid size from D0 to D30 (n=15; 5 organoids in triplicates were pooled together for quantification). Organoid area is shown in mm². Error bars represent SD.

(D) WB of D15 WA01 TH midbrain organoids expressing the DN marker, TH, and midbrain progenitor marker, FOXA2. GAPDH was used as loading control.

(E) qRT-PCR showing mRNA expression of midbrain progenitor markers FOXA2 and LMX1A in D15 midbrain organoids as a fold change over hPSCs (n=3). ACTB was used as a housekeeping gene for normalization. Error bars represent SD.

(F) Fluorescent image of D20 WA01 TH midbrain organoids expressing the TH-TdTomato reporter. Scale bar: 250 μm.